Class BI and CI scavenger receptors

ABSTRACT

Two distinct scavenger receptor type proteins having high affinity for modified lipoproteins and other ligands have been isolated, characterized and cloned. HaSR-BI, an AcLDL and LDL binding scavenger receptor, which is distinct from the type I and type II macrophage scavenger receptors, has been isolated and characterized and DNA encoding the receptor cloned from a variant of Chinese Hamster Ovary Cells, designated Var-261. dSR-CI, a non-mammalian AcLDL binding scavenger receptor having high ligand affinity and broad specificity, was isolated from  Drosophila melanogaster . The isolated receptors are useful in screening for drugs that inhibit uptake of cholesterol in endothelial or adipose cells or macrophages, respectively. They are also useful as probes for the isolation of other lipoprotein receptors and in research the roles of these receptors.

BACKGROUND OF THE INVENTION

The present invention is generally in the area of new scavenger receptor proteins present on cells which can mediate lipid or lipoprotein uptake, genes encoding these proteins and methods of detection and use thereof.

The intercellular transport of lipids through the circulatory system requires the packaging of these hydrophobic molecules into water-soluble carriers, called lipoproteins, and the regulated targeting of these lipoproteins to appropriate tissues by receptor-mediated endocytic pathways. The most well characterized lipoprotein receptor is the LDL receptor, which binds to apolipoproteins B-100 (apoB-100) and E (apoE), which are constituents of low density lipoprotein, the principal cholesteryl-ester transporter in human plasma (LDL), very low-density lipoprotein, a triglyceride-rich carrier synthesized by the liver (VLDL), intermediate-density lipoprotein (IDL), and catabolized chylomicrons (dietary triglyceride-rich carriers synthesized by the liver).

All members of the LDL receptor gene family consist of the same basic structural motifs, shown in FIG. 1A. Ligand-binding (complement-type) cysteine-rich repeats of approximately 40 amino acids are arranged in clusters (ligand-binding domains) that contain between two and eleven repeats. Ligand-binding domains are always followed by EGF-precursor homologous domains. In these domains, two EGF-like repeats are separated from a third EGF-repeat by a spacer region containing the YWTD motif. In LRP and gp330, EGF-precursor homologous domains are either followed by another ligand-binding domain or by a spacer region. The EGF-precursor homology domain, which precedes the plasma membrane, is separated from the single membrane-spanning segment either by an O-linked sugar domain (in the LDL receptor and VLDL receptor) or by one (in C. elegans and gp330) or six EGF-repeats (in LRP). The cytoplasmic tails contain between one and three “NPXY” internalization signals required for clustering of the receptors in coated pits. In a later compartment of the secretory pathway, LRP is cleaved within the eighth EGF-precursor homology domain. The two subunits LRP-515 and LRP-85 (indicated by the brackets) remain tightly and non-covalently associated. Only partial amino acid sequence of the vitellogenin receptor and of gp330 are available.

LDL receptors and most other mammalian cell-surface receptors that mediate endocytosis, adhesion, or signaling exhibit two common ligand-binding characteristics: high affinity and narrow specificity. However, two additional lipoprotein receptors have been identified which are characterized by high affinity and broad specificity: the macrophage scavenger receptors type I and type II.

Scavenger receptors mediate the endocytosis of chemically modified lipoproteins, such as acetylated LDL (AcLDL) and oxidized LDL (OxLDL), and have been implicated in the pathogenesis of atherosclerosis (Krieger and Herz, 1994 J. Annu. Rev. Biochem. 63, 601-637; Brown and Goldstein, 1983 Annu. Rev. Biochem. 52, 223-261; Steinberg et al., 1989 N. Engl. J. Med. 320, 915-924). Macrophage scavenger receptors exhibit complex binding properties, including inhibition by a wide variety of polyanions, such as maleylated BSA (M-BSA) and certain polynucleotides and polysaccharides, as well as unusual ligand-cross competition (Freeman et al., 1991 Proc. Natl. Acad. Sci. U.S.A. 88, 4931-4935, Krieger and Herz, 1994). Several investigators have suggested that there may be at least three different classes of such receptors expressed on mammalian macrophages, including receptors which recognize either AcLDL or OxLDL, or both of these ligands (Sparrow et al., 1989 J. Biol. Chem. 264, 2599-2604; Arai et al., 1989 Biochem. Biophys. Res. Commun. 159, 1375-1382; Nagelkerke et al., 1983 J. Biol. Chem. 258, 12221-12227).

The first macrophage scavenger receptors to be purified and cloned were the mammalian type I and II receptors. These are trimeric integral membrane glycoproteins whose extracellular domains have been predicted to include a-helical coiled-coil, collagenous and globular structures (Kodama et al., 1990 Nature 343, 531-535; Rohrer et al., 1990; Krieger and Herz, 1994). The collagenous domain, shared by the type I and type II receptors, apparently mediates the binding of polyanionic ligands (Acton et al., 1993 J. Biol. Chem. 268, 3530-3537; Doi et al., 1993 J. Biol. Chem. 268, 2126-2133). The type I and type II molecules, which are the products of alternative splicing of a single gene, are hereafter designated class A scavenger receptors (SR-AI and SR-AII). The class A receptors, which bind both AcLDL and OxLDL (Freeman et al., 1991), have been proposed to be involved in host defense and cell adhesion, as well as atherogenesis (Freeman et al., 1991; Krieger, 1992 Trends Biochem. Sci. 17, 141-146; Fraser et al., 1993 Nature 364, 343-346; Krieger and Herz, 1994).

Models of the predicted quaternary structures of the type I and type II macrophage scavenger receptors are shown in FIG. 1B. Both contain six domains, of which the first five are identical: the N-terminal cytoplasmic region, the transmembrane region, spacer, α-helical coil, and collagen-like domains. The C-terminal sixth domain of the type I receptor is composed of an eight-residue spacer followed by a 102-amino acid cysteine-rich domain (SRCR), while the sixth domain of the type II receptor is only a short oligopeptide.

Using a murine macrophage cDNA library and a COS cell expression cloning technique, Endemann, Stanton and colleagues, (Endemann, et al. 1993 J. Biol. Chem. 268, 11811-11816; Stanton, et al. J. Biol. Chem. 267, 22446-22451), reported the cloning of cDNAs encoding two additional proteins that can bind OxLDL. The binding of OxLDL to these proteins was not inhibited by AcLDL. These proteins are FcgRII-B2 (an Fc receptor) (Stanton et al., 1992) and CD36 (Endemann et al., 1993). The significance of the binding of OxLDL to FcgRII-B2 in transfected COS cells is unclear because FcgRII-B2 in macrophages apparently does not contribute significantly to OxLDL binding (Stanton et al., 1992). However, CD36 may play a quantitatively significant role in OxLDL binding by macrophages (Endemann et al., 1993). In addition to binding oxidized LDL, CD36 binds thrombospondin (Asch et al., 1987 J. Clin. Invest. 79, 1054-1061), collagen (Tandon et al., 1989 J. Biol. Chem. 264, 7576-7583), long-chain fatty acids (Abumrad et al., 1993 J. Biol. Chem. 268, 17665-17668) and Plasmodium falciparum infected erythrocytes (Oquendo et al., 1989 Cell 58, 95-101). CD36 is expressed in a variety of tissues, including adipose, and in macrophages, epithelial cells, monocytes, endothelial cells, platelets, and a wide variety of cultured lines (Abumrad et al., 1993; and see Greenwalt et al., 1992 Blood 80, 1105-1115 for review). Although the physiologic functions of CD36 are not known, it may serve as an adhesion molecule due to its collagen-binding properties. It is also been proposed to be a long-chain fatty acid transporter (Abumrad et al., 1993) and a signal transduction molecule (Ockenhouse et al., 1989 J. Clin. Invest. 84, 468-475; Huang et al., 1991), and may serve as a receptor on macrophages for senescent neutrophils (Savill et al., 1991 Chest 99, 7 (suppl)).

Modified lipoprotein scavenger receptor activity has also been observed in endothelial cells (Arai et al., 1989; Nagelkerke et al., 1983; Brown and Goldstein, 1983; Goldstein et al., 1979 Proc. Natl. Acad. Sci. U.S.A. 76, 333-337). The endothelial cell activity apparently is not mediated by the class A scavenger receptors (Bickel et al., 1992 J. Clin. Invest. 90, 1450-1457; Arai et al., 1989; Nagelkerke et al., 1983; Via et al., 1992 The Faseb J. 6, A371), which are expressed almost exclusively by macrophages (Naito et al., 1991 Am. J. Pathol. 139, 1411-1423; Krieger and Herz, 1994). In vivo and in vitro studies suggest that there may be scavenger receptor genes expressed in endothelial cells and macrophages which differ from both the class A scavenger receptors and CD36 (Haberland et al., 1986 J. Clin. Inves. 77, 681-689; Via et al., 1992; Sparrow et al., 1989; Horiuchi et al., 1985 J. Biol. Chem. 259, 53-56; Arai et al., 1989; and see below). Via, Dressel and colleagues (Ottnad et al., 1992 Biochem J. 281, 745-751) and Schnitzer et al. 1992 J. Biol. Chem. 267, 24544-24553) have detected scavenger receptor-like binding by relatively small membrane associated proteins of 15-86 kD. In addition, the LDL receptor related protein (LRP) has been shown to bind lipoprotein remnant particles and a wide variety of other macromolecules. Both the mRNA encoding and the LRP protein are found in many tissues and cell types (Herz, et al., 1988 EMBO J. 7:4119-4127; Moestrup, et al., 1992 Cell Tissue Res. 269:375-382), primarily the liver, the brain and the placenta. The predicted protein sequence of the LRP, shown in FIG. 1A, consists of a series of distinctive domains or structural motifs, which are also found in the LDL receptor.

Based on the information known regarding the structures and functions of multiligand lipoprotein receptors present on macrophages, it would clearly be of benefit to isolate and clone other members of the lipoprotein receptor family present on macrophages, especially from non-mammalian species, in order to investigate which aspects of these molecules are most conserved and which portions can therefore be selectively targeted for stimulation or inhibition of binding, and on other cell types, the structure and function of whose receptors are not characterized.

It is therefore an object of the present invention to provide the structure, amino acid sequence, and DNA sequence encoding a previously undescribed lipoprotein receptor present on mammalian cells.

It is another object of the present invention to provide the structure, amino acid sequence, and DNA sequence encoding a lipoprotein receptor present on insect macrophages.

It is a further object of the present invention to provide methods and reagents for use in isolating and characterizing lipoprotein receptors that are not type I and type II macrophage scavenger receptors nor classic LDL receptors.

It is yet a still further object of the present invention to provide methods and reagents for designing drugs that can stimulate or inhibit the binding of lipoprotein receptors that are not type I and type II macrophage scavenger receptors nor classic LDL receptors.

SUMMARY OF THE INVENTION

Two distinct scavenger receptor type proteins having high affinity for modified lipoproteins and other ligands have been isolated, characterized and cloned. HaSR-BI, an AcLDL and LDL binding scavenger receptor, which is distinct from the type I and type II macrophage scavenger receptors, has been isolated and characterized and DNA encoding the receptor cloned from a variant of Chinese Hamster Ovary Cells, designated Var-261. dSR-CI, a non-mammalian AcLDL binding scavenger receptor having high ligand affinity and broad specificity, was isolated from Drosophila melanogaster.

The presence of scavenger receptors on both mammalian and Drosophila macrophages indicates that they mediate critical, well-conserved functions, including pathogen recognition, and that they may have appeared early in the evolution of host defense systems. In this regard, it is known that postembryonic macrophagelike hemocytes in Drosophila participate in wound healing, encapsulation of pathogens, and phagocytosis. Due to the known association between atherosclerosis and macrophages, and the uptake of cholesterol by macrophages which is mediated by scavenger receptor proteins, the isolated receptors are useful in screening for drugs that inhibit uptake of cholesterol by cells expressing these receptors. They are also useful as probes for the isolation of other lipoprotein receptors and in research of the roles of these receptors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic of the structure of the LDL receptor family members, as described by the prior art discussed in the Background of the Invention.

FIG. 1B is a schematic of the structure of the macrophage scavenger receptors, type I and type II, discussed in the Background of the Invention.

FIGS. 2A, 2B, and 2C are graphs of the concentration dependence of ¹²⁵I-AcLDL interaction with Var-261 and ldlA-7 cells at 4° C. and 37° C. FIG. 2A is binding at 4° C.; FIG. 2B is binding plus uptake at 37° C.; and FIG. 2C is degradation at 37° C., measured as ng ¹²⁵I-AcLDL/5 hr/mg cell protein versus ¹²⁵I-AcLDL (μg protein/ml).

FIGS. 3A and 3B are graphs of the binding of ¹²⁵I-AcLDL to haSR-BI expressed in transfected COS cells (FIG. 3A) and specificity of binding of ¹²⁵I-AcLDL to Var-261 and ldlA[haSR-BI] (FIG. 3B) measured as the percent of control binding in the presence of competitor: M-BSA (10 μg/ml), poly G (500 μg/ml), Fucoidin (200 μg/ml), Carrageenan (200 μg/ml), and LDL (500 μg/ml).

FIGS. 4A and 4B are graphs of the binding of ¹²⁵I-AcLDL to CD36 expressed in transfected COS cells (FIG. 4A) and specificity of binding of ¹²⁵I-AcLDL to transfected COS cells (FIG. 4B) measured as percent of control in the presence of a competitor: M-BSA (4 μg/ml), AcLDL (500 μg/ml), Fucoidin (200 μg/ml), Poly I (500 μg/ml), Poly G (500 μg/ml), and ReLPS (250 μg/ml).

FIG. 5 is a graph of lipoprotein inhibition of ¹²⁵I-AcLDL binding to haSR-BI and huCD36, measured as percent of control binding at 4° C., to either COS[haSR-BI] or COS[huCD36], alone, in the presence of AcLDL, OxLDL or LDL.

FIGS. 6A, 6B, and 6C are graphs of the concentration dependent binding, uptake and degradation of ¹²⁵I-AcLDL to CHO[dSR-CI]-2.6a cells: FIG. 6A is binding at 4° C.; FIG. 6B is binding plus uptake at 37° C.; and FIG. 6C is degradation at 37° C., measured as ng ¹²⁵I-AcLDL/5 hr/mg cell protein versus ¹²⁵I-AcLDL (μg protein/ml).

DETAILED DESCRIPTION OF THE INVENTION

In order to isolate, characterize, and clone the genes for new scavenger receptors and other lipoprotein receptors, cDNA and genomic libraries are prepared from cells in which activities have been identified which are characteristic of scavenger receptors: binding to lipoproteins such as LDL, AcLDL and/or oxidized-LDL; hybridization screening of the genomic libraries using probes generated from the nucleic acid sequences of cloned receptors; expression cloning by transient expression in COS cells and/or expression cloning by stable expression in CHO cells; analysis of the cloned cDNA: verification, sequencing and sequence analysis; identification and isolation of the genomic DNA including regulatory sequences; immunochemical analysis of the structure and biosynthesis of the new scavenger receptors; and characterization of the binding properties of the new receptors and comparisons with type I and type II receptors.

Isolation and Characterization of a Mammalian Scavenger Receptor Protein on CHO Cells.

To extend the analysis of the structure and function of mammalian modified lipoprotein scavenger receptors, a variant Chinese hamster ovary cell line, Var-261, which, based on ligand specificity, expresses an apparently novel polyanion binding scavenger receptor, was identified and characterized. The cDNA for a scavenger receptor, haSR-BI, which is a new member of the CD36 family of membrane proteins (class B scavenger receptors), was isolated from the Var-261 cells. Although isolated from the same cells, haSR-BI is not responsible for the novel polyanion binding receptor activity of Var-261 cells.

The ligand specificities of CD36 and haSR-BI expressed in transfected cell lines was compared with that of Var-261 cells. haSR-BI differs from CD36 and other modified lipoprotein receptors described to date in that its binding of AcLDL is inhibited by native LDL.

The cDNA encoding SR-BI yields a predicted protein sequence of 509 amino acids which is approximately 30% identical to those of the three previously identified CD36 family members. Northern blot analysis of murine tissues shows that SR-BI is most abundantly expressed in fat and is present at moderate levels in lung and liver. Furthermore, SR-BI mRNA expression is induced upon differentiation of 3T3-L1 cells into adipocytes. Both SR-BI and CD36 display high affinity binding for acetylated LDL with an apparent dissociation constant in the range of approximately 5 μg protein/ml. The ligand binding specificities of CD36 and SR-BI, determined by competition assays, are similar, but not identical: both bind modified proteins (acetylated LDL, maleylated BSA), but not the broad array of other polyanions (e.g. fucoidin, polyinosinic acid, polyguanosinic acid) which are ligands of the class A receptors. Native LDL, which does not compete for the binding of acetylated LDL to either class A receptors, CD36 or Var-261 cells, unexpectedly competes for binding to SR-BI. SR-BI and CD36 therefore define a second class of scavenger receptors, designated class B, which are defined as members of the CD36 family which can bind to modified LDL. The ability of other known members of the CD36 family to bind to modified LDLs has not been reported. Class B scavenger receptors may play a role in the in vivo and in vitro uptake of modified proteins previously described by Haberland et al., 1989 J. Immunol. 142, 855-862; Villaschi et al., 1986 Microvasc. Res. 32, 190-199; Horiuchi et al., 1985; Predescu et al., 1988 J. Cell Biol. 107, 1729-1738).

These methods and conclusions are described in greater detail below. Abbreviations: LDL (low density lipoprotein), OxLDL (oxidized LDL), AcLDL (acetylated LDL), M-BSA (maleylated BSA), CHO (Chinese hamster ovary), haSR-BI (hamster scavenger receptor type BI), mSR-AII (murine scavenger receptor type AII), huCD36 (human CD36).

Materials and Methods

Materials

LDL, AcLDL, ¹²⁵I-labeled AcLDL (100-400 cpm/ng protein), and newborn calf and human lipoprotein-deficient sera were prepared as described by Goldstein et al., 1983 Methods Enzymol. 98, 241-260; Krieger, 1983 Cell 33, 413-422. For some preparations of LDL and AcLDL, the following additional precautions were taken to prevent inadvertent oxidation: 1) 20 μM β-hydroxytoluene was added to the plasma before separation of the lipoproteins on the gradient, 2) one hour prior to all dialysis steps the dialysis solutions were degassed by vacuum, followed by purging with nitrogen gas 3) whenever possible purification procedures were performed in the dark or low light, and 4) the preparations were stored in the dark under argon. OxLDL was prepared by dialyzing 1 ml of LDL (4-10 mg/ml) (prepared without β-hydroxytoluene) against saline solution containing 5 μM Cu₂SO₄ (2×500 ml) for 24 to 48 hours at 4° C. Using a lipid peroxidation assay (El-Saadani et al., 1989), very little inadvertent oxidation in LDL and AcLDL preparations isolated with or without the precautions listed above was measured. Polyriboguanylic acid (poly G), polyriboinosinic acid (poly I), fucoidin, dextran sulfate, chondroitin sulfate, ReLPS (S. minnesota Re-595) and carrageenan (type III Kappa) were obtained from Sigma Chemical Co. Maleylated BSA (M-BSA) was prepared as described by Goldstein et al., 1979. The CD36 expression vector was a gift from B. Seed (Massachusetts General Hospital, Boston). 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) and 3-pyrenemethyl-23,24-dinor-5-cholen-22-oate-3b-yl oleate (PMCAO) were obtained from Molecular Probes (Eugene, Oreg.) and were used to prepare fluorescently labeled AcLDL as described by Krieger et al., 1979 J. Supra. Struct. 10, 467-478; Krieger, 1986 Meth. Enzymol. 128, 608-613; Pitas et al., 1985 J. Cell. Biol. 100, 103-117; Kingsley and Krieger, 1984 Proc Natl. Acad. Sci. USA 88, 7844-7848. Compactin and dioctadecylamidoglycylspermine (DOGS) were generous gifts from A. Endo and J. R. Falck, respectively.

Cell Culture and Transfections

CHO, ldlA (clone 7) and COS M6 cells were grown in culture as described by Krieger et al., 1983 Proc. Natl. Acad. Sci. USA 80, 5607-5611; Acton et al., 1993. All incubations with cells were performed at 37° C. in a humidified 5% CO₂/95% air incubator unless otherwise noted. COS M6 cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mM glutamine (medium A). For transient transfections other than in the expression cloning experiments, 1×10⁶ COS cells were plated in 100 mm dishes in medium A on day 0. On day 1, plasmid DNA (between 2 and 10 μg in 1.9 ml phosphate buffered saline [PBS, calcium/magnesium free]) was mixed with 100 μl of 10 mg/ml DEAE-dextran (Pharmacia) and then added to each dish, which was then incubated at 37° C. with gentle shaking every 10 minutes. After 30 minutes, 8 ml of medium A supplemented with 80 μM chloroquine (Sigma) were added and the cells were incubated for another 2.5 hrs. The medium was removed and the cells were shocked with 5 ml of 10% (v/v) dimethylsulfoxide in medium A for 2.5 minutes. This medium was quickly removed by aspiration, the cells were washed with 10 ml of PBS (containing calcium and magnesium), and 10 ml of fresh medium A were added. On day 2 the cells were harvested with trypsin and replated (0.5-1×10⁶ cells/well in 6-well dishes) in medium A containing 1 mM sodium butyrate. On day 3, the cells were assayed for ligand binding at 4° C. and for binding plus uptake (binding/uptake) and degradation at 37° C. Stable transfections were performed as follows: ldlA cells were plated at 1×10⁶ cells per 100 mm dish in medium B (Ham's F12 containing 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mM glutamine) supplemented with 10% fetal calf serum. On day 2, the cells were washed three times with medium B. A complex of DNA and DOGS (dioctadecylamidoglycylspermine, which was a gift from J. R. Falck) was prepared by adding 150 μl of a DNA solution (12 μg of plasmid and 1.2 μg of supercoiled pSV2neo in 300 mM NaCl ) to 150 μl of solution A (0.33 mg/ml DOGS, 300 mM NaCl). The DNA/DOGS complex was added to the cells in 3.6 ml of serum-free Opti-MEM medium (GIBCO) containing penicillin (100 U/ml) and streptomycin (100 μg/ml). After a 14 hr transfection period, the cells were washed three times with medium B and incubated in medium B plus 10% fetal calf serum for one day before being replated at a density of 3×10⁶ cells per 100 mm dish. One day later, the culture medium was replaced with selection medium, medium B supplemented with 10% fetal calf serum and 0.25 mg/ml G418. This medium was changed every two to three days, and G418-resistant colonies appeared 10 to 12 days after transfection. The colonies were screened for DiI-AcLDL endocytosis and positive colonies were picked and grown to mass culture for further analysis.

Isolation of Var-261 Cells

Var-261 cells, a rare spontaneously arising variant expressing a novel scavenger receptor activity, were isolated during a transfection/selection experiment as follows. Human liver genomic DNA prepared as described by Sege et al., 1984 Nature 307, 742-745, was transfected into ldlA (clone 7) cells, a CHO cell mutant clone whose defective LDL receptor gene results in an essentially LDL receptor-negative phenotype (Sege, et al., 1986 Mol. Cell. Biol. 6, 3268-3277; Kingsley et al., 1986 Mol. Cell. Biol. 6, 2734-2737; Kingsley and Krieger, 1984). After transfection, variants expressing scavenger receptor activity were isolated by growing the cells in MAC selection medium (Penman et al., 1991 J. Biol. Chem. 266, 23985-23993) [250 μM Mevalonate, 3 μg protein/ml of AcLDL, 40 μM Compactin, and 3% (v/v) newborn calf lipoprotein-deficient serum in medium B. In MAC medium, endogenous cholesterol synthesis is inhibited by compactin and only those cells that are able to obtain cholesterol by the endocytosis of AcLDL can survive. The parental ldlA cells used for the transfection cannot grow in MAC medium. After incubation in MAC medium for 29 days, surviving colonies were refed with medium B supplemented with 3% (v/v) newborn calf lipoprotein-deficient serum containing AcLDL which was fluorescently labeled by reconstitution of the lipid core (Krieger, 1986) with the pyrene-based lipophilic dye PMCAO (Krieger et al., 1979). Colonies which accumulated significant amounts of fluorescence from the lipoprotein, as determined by fluorescence microscopy (Krieger et al., 1981 J. Mol. Biol. 150, 167-184), were harvested and maintained in MAC selection medium. One colony, designated Var-261, exhibited significant levels of AcLDL binding, uptake and degradation activity which were dramatically greater than those in either the parental ldlA cells or any of the other colonies. Southern blot analysis using human genomic DNA as a probe showed that there was no detectable human-specific repeat DNA in Var-261 cells. Thus, Var-261 cells are presumably rare, spontaneously arising variants which express an endogenous hamster scavenger receptor gene. This expression could be due to the activation of an otherwise silent gene. Alternatively, it might be due to a spontaneous mutation which conferred novel activity on an endogenous protein with some other function (e.g., see Faust and Krieger, 1987 J. Biol. Chem. 262, 1996-2004, and Chen et al., 1990 J. Biol. Chem. 265, 3116-3123).

¹²⁵I-AcLDL Binding, Uptake and Degradation Assays

Scavenger receptor activities at 37° C. were measured by ligand binding, uptake and degradation assays as described by Krieger, 1983; Freeman et al., 1991). Although binding and uptake at 37° C. were determined separately using dextran-sulfate to free surface bound lipoproteins from the cells as described for LDL receptor assays by Goldstein et al., 1983; Basu et al., 1983 Science 219, 871-873), this method for separating bound from internalized ligand for ¹²⁵I-AcLDL and scavenger receptors has not been independently validated. Accordingly, the values for binding and uptake were combined and are presented as binding plus uptake observed after a 5 hour incubation and are expressed as ng of ¹²⁵I-AcLDL protein per 5 hr per mg cell protein. Degradation activity is expressed as ng of ¹²⁵I-AcLDL protein degraded in 5 hours per mg of cell protein. The specific, high affinity values presented represent the differences between the results obtained in the presence (single determinations) and absence (duplicate determinations) of excess unlabeled competing ligand (75 to 200 μg/ml of M-BSA). Cell surface 4° C. binding was assayed using either method A or method B as indicated. In method A, cells were prechilled on ice for 15 min, refed with ¹²⁵I-AcLDL in ice-cold medium B supplemented with 10% (v/v) fetal bovine serum, with or without 75-200 μg/ml unlabeled M-BSA, and incubated 2 hr at 4° C. on a shaker. Cells were then washed rapidly three times with Tris wash buffer (50 mM Tris-HCl, 0.15 M NaCl, pH 7.4) containing 2 mg/ml BSA, followed by two 5 min washes, and two rapid washes with Tris wash buffer without BSA. The cells were solubilized in 1 ml of 0.1 N NaOH for 20 min at room temperature on a shaker, 30 μl were removed for protein determination, and the radioactivity in the remainder was determined using a LKB gamma counter. Method B differed from method A in that the cells were prechilled for 45 minutes, the medium contained 10 mM HEPES and 5% (v/v) human lipoprotein-deficient serum rather than fetal bovine serum, and the cell-associated radioactivity released by treatment with dextran sulfate was measured as described by Krieger, 1983; Freeman et al., 1991).

Preparation of Var-261 cDNA Library

Poly A⁺mRNA was isolated from Var-261 cells using standard procedures (Sambrook, Fritsch, and Maniatis. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory Press (1989)). This mRNA was used to synthesize double-stranded cDNA using a Not I unidirectional primer (Invitrogen) according to the method of Aruffo and Seed, 1987 Immunology 84, 8573-8577. BstXI linkers (Invitrogen) were added to the cDNA. The isolated cDNA was digested with Not I, size selected from agarose gels (in three size groups: 1 to 2 kb, 2 to 4 kb, greater than 4 kb), and ligated into the expression vector pcDNA I (Invitrogen). The average insert size for the entire Var-261 unidirectional library was approximately 1.5 kb. The DNAs for transfection experiments were prepared as follows. E. coli strain MC1061/P3 was transformed with the cDNA expression plasmids by electroporation and the transformed cells were plated on LB-A/T plates (Luria broth with ampicillin (15 μg/ml)/tetracycline (8 μg/ml)) (Sigma) to obtain isolated colonies at densities of approximately 5,000 per 150 mm dish. Each plate, representing one pool, was scraped to recover the bacteria and a pool of library plasmids was isolated from each bacterial mixture using a midiprep method (miniprep method scaled up 5 to 10 fold (Sambrook et al., 1989)).

Expression Cloning by Transient Expression in COS Cells

On day 0, COS M6 cells were plated in 35 mm culture dishes (3 to 4×10⁵ cells/dish) in medium A. On day 1, the cells in each dish were transfected with 0.5 μg/dish of expression library DNA from a single pool following the DEAE-dextran method of Cullen 1987 Methods in Enz. 152, 684-704. On day 2, monolayers were refed with medium A containing 1 mM sodium butyrate (modified medium A). On day 3, the monolayers were refed with modified medium A containing one to five μg protein/ml of DiI-labeled AcLDL (DiI-AcLDL). After a 5 hr incubation at 37° C., the plates were washed two times with PBS and the cells were fixed with 3.7% formaldehyde in PBS for 10 min at room temperature. The presence of fluorescent DiI in the fixed cells was determined by visual inspection using a Leitz inverted fluorescence microscope with a rhodamine filter package as described by Kingsley and Krieger, (1984). Each transfection experiment included negative control transfections with empty vector pcDNA I (0 to 20 DiI-positive cells per dish) and positive control transfections with a mixture of a pcDNA I-based bovine type II macrophage scavenger receptor expression vector, pXSR3 (Rohrer et al., 1990) and the empty vector pcDNA I (1:5000 ratio, 200 to 400 DiI-positive cells per dish). A positive pool was serially subdivided and retested to permit the purification of the single positive plasmid, phaSR-BI.

Northern blot analysis

0.5 micrograms of poly(A)+ RNA prepared from different murine tissues or from 3T3-L1 cells on zero, two, four, six or eight days after initiation of differentiation into adipocytes as described by Baldini et al., 1992 Proc. Natl. Acad. Sci. U.S.A. 89, 5049-5052, was fractionated on a formaldehyde/agarose gel (1.0%) and then blotted and fixed onto a Biotrans™ nylon membrane. The blots were hybridized with the indicated probes that were ³²P-labeled (2×10⁶ dpm/ml, random-primed labeling system). The hybridization and washing conditions, at 42° C. and 50° C., respectively, were performed as described by Charron et al., 1989 Proc. Natl. Acad. Sci. U.S.A. 86, 2535-2539. The probe for haSR-BI mRNA analysis was a 0.6 kb BamHI fragment from the cDNA's coding region. The coding region of murine cytosolic hsp70 gene (Hunt and Calderwood, 1990 Gene 87, 199-204) was used as a control probe for equal mRNA loading.

Other Procedures

Protein concentrations were determined by the method of Lowry et al. 1951 J. Biol. Chem. 193, 265-275. DNA sequencing was performed using Sequenase 2.0 kit according to manufacturers instructions and the reported results for the coding region were verified by determining the sequence from both strands of the template. Oligonucleotide primers were prepared in the MIT Biopolymers Laboratory. Polymerase chain reaction (PCR) was used to generate a fragment of the hamster class A scavenger receptor DNA as follows. Primers based on the sequence of one exon of the murine scavenger receptor gene (Ashkenas et al., 1993 J. Lipid Res. 34, 983-1000) (5′AATGAAGAACTGCTTAGTTT 3′ (Sequence ID No. 1) and 5′ AATCAAGGAATTTAACTG 3′ (Sequence ID No. 2)) were used to amplify a fragment of the expected size (240 base pairs) from hamster ldlA cell genomic DNA. This amplicon was cloned into pCR1000 (Invitrogen) to generate the plasmid pSA1, which was sequenced.

Results

Isolation and Characterization of Variant Var-261

High Affinity Binding, Uptake and Degradation of AcLDL

The LDL receptor-deficient CHO cell line ldlA was processed and subjected to nutritional selection for rare variants which expressed endocytic receptors for AcLDL. One of the isolates, designated Var-261, was examined further. Var-261 and ldlA cells were plated on day 0 at 350,000 and 250,000 cells/well, respectively, in 6-well dishes in medium B plus 3% (v/v) newborn calf lipoprotein-deficient serum, and assayed on day 2 as described above. ¹²⁵I-AcLDL binding at 4° C. for 2 hrs was measured using method B. ¹²⁵I-AcLDL binding plus uptake and degradation at 37° C. was measured after a 5 hr incubation. The high affinity values shown represent the differences between measurements made in the absence (duplicate incubations) and presence (single incubations) of an excess of the unlabeled competitor M-BSA (100 μg/ml).

As shown by FIG. 2A, these cells exhibited high affinity, saturable ¹²⁵I-AcLDL binding at 4° C. The binding at 37° C. and subsequent uptake shown in FIG. 2B and lysosomal degradation shown in FIG. 2C were characteristic of receptor-mediated endocytosis. Degradation, but not binding plus uptake, at 37° C. was inhibited by the lysosomotrophic drug chloroquine. In contrast, there was virtually no high affinity binding, uptake, or degradation of ¹²⁵I-AcLDL by the parental ldlA cells or wild-type CHO cells. The scavenger receptor activity in Var-261 cells was not suppressed by the addition of sterols to the medium.

Broad Polyanion Binding Specificity

A hallmark of macrophage scavenger receptors is their broad polyanion binding specificity, usually assessed by competition with polyanionic ligands for ¹²⁵I-AcLDL binding, uptake and degradation. For further characterization, the ligand specificity of the scavenger receptor activity of Var-261 cells was compared with that of transfected COS cells transiently expressing the murine type II macrophage scavenger receptor, a class A scavenger receptor (mSR-AII).

COS cells were transfected on day 1 with murine scavenger receptor type II. On day 2 the transfected COS cells and Var-261 cells were set in 6-well dishes (1×10⁶ and 0.5×10⁶cells/well, respectively). On day 3, ¹²⁵I-AcLDL (5 μg/ml) in medium B plus 10% fetal bovine serum was added to the cells and degradation was measured after a 5 hr incubation at 37° C. in the absence (duplicate determinations) or presence (duplicate determinations) of the indicated amounts of polyanions.

The classic scavenger receptor polyanionic ligands AcLDL, fucoidin, and poly G were all effective competitors of ¹²⁵I-AcLDL degradation by both Var-261 and the transfected COS cells, and native LDL did not inhibit the activity in either cell type. Nevertheless, the binding specificity in Var-261 cells differed from that in macrophages and for class A receptors in several critical ways. Poly I, a very effective competitor of class A scavenger receptors (greater than 50% inhibition at 2.5 μg/ml) only partially inhibited the activity in Var-261 cells (34% inhibition at 400 μg/ml). In contrast, maleylated BSA (M-BSA) inhibited the scavenger receptor activity in Var-261 cells at concentrations far lower that those required for inhibition of the murine type II receptors. The ReLPS form of endotoxin, which is a scavenger receptor competitor exhibiting complex binding properties for class A scavenger receptors (Ashkenas et al., 1993), only partly inhibited the activity in Var-261 cells at the high concentration of 250 μg/ml.

Determination if Hamster Class A Scavenger Receptor DNA is Expressed in Var-261 Cells

The striking differences in ligand specificities between the scavenger receptor activity in Var-261 cells and those exhibited by the murine and bovine class A scavenger receptors (Ashkenas et al., 1993; Kodama et al., 1990; Rohrer et al., 1990) strongly suggested that the Var-261 cells did not express class A receptors. However, there was a possibility that the novel specificity was merely a consequence of species differences (e.g., hamster-derived Var-261 cells vs. murine SR-AII). To determine if this was the case, a 240 base pair fragment of the hamster class A macrophage scavenger receptor gene was isolated by polymerase chain reaction (PCR) using genomic CHO cell DNA as a template. This hamster gene fragment, which is found in both type I and II macrophage scavenger receptors, was cloned and sequenced. This fragment has between 75%-85% -nucleotide sequence identity with its human, murine, rabbit and bovine counterparts (Kodama et al., 1990; Rohrer et al., 1990; Ashkenas et al., 1993; Matsumoto et al., 1990 Proc. Natl. Acad. Sci. USA 87, 9133-9137; Bickel and Freeman, 1992) and, with the exception of the amino acid corresponding to murine position 117, was consistent with the previously identified consensus sequence defined for residues 95-159 (Ashkenas et al, 1993).

This hamster fragment was used as a probe in Northern analysis of Var-261 cell mRNA. Under conditions where the hamster probe recognized murine scavenger receptor mRNA from CHO cells transfected with the murine scavenger receptor cDNA (Ashkenas et al., 1993), no signal was detected in the hamster-derived Var-261 or control CHO cell mRNA, even after extensive overexposure of the film. Thus, Var-261 cells do not express significant amounts of the hamster type I or II class A receptors and must instead express a different scavenger receptor.

Cloning of haSR-BI Receptor cDNA from Var-261

In an attempt to clone a cDNA encoding this apparently new scavenger receptor, a cDNA expression library was prepared from Var-261 poly A⁺ mRNA, the library was divided into small pools (approximately 5000 clones/pool), the pools were transfected into COS cells and the transiently transfected cells visually screened for endocytosis of fluorescent AcLDL (DiI-AcLDL). A receptor-positive pool was obtained after screening approximately 450,000 clones and this pool was subdivided repeatedly until a single functional plasmid (designated phaSR-BI for plasmid encoding hamster scavenger receptor type BI) was obtained.

FIGS. 3A and 3B are graphs of ¹²⁵I-AcLDL binding to haSR-BI expressed in transfected COS cells: FIG. 3A demonstrates the concentration dependence in COS[haSR-BI] cells and FIG. 3B demonstrates the ligand specificity in ldlA[haSR-BI] cells. To measure binding of AcLDL, on day 1, COS cells were transfected with the expression vector for the hamster scavenger receptor type BI (haSR-BI). On day 2 the transfected COS cells were plated in 6-well dishes (1×10⁶ cells/well in medium A plus 1 mM sodium butyrate). On day 3 between 0 and 50/g/ml of ¹²⁵I-AcLDL were added to the monolayers at 4° C. and binding was measured using Method A. The high affinity values shown in the Figures represent the differences between measurements made in the absence (duplicate incubations) and presence (single incubations) of an excess of the unlabeled competitor M-BSA (200 μg/ml). To measure binding specificity, on day 1, Var-261 and ldlA[haSR-BI] cells were plated in 6-well dishes (0.2×10⁶ cells/well in MAC medium or medium B containing 10% fetal bovine serum and 0.25 mg/ml G418, respectively). On day 3 the binding of ¹²⁵I-AcLDL (5 μg/ml) at 4° C. in the absence or presence of the indicated competitors was measured using Method A. The values shown in the Figures represent the averages of duplicate determinations. The 100% of control values for Var-261 and ldlA[haSR-BI] were 1809 and 293 ng/mg cell protein, respectively.

The haSR-BI plasmid conferred high affinity (K_(d)(4° C.) approximately 5 μg protein/ml) ¹²⁵I-AcLDL binding on transiently transfected COS cells, as shown by FIG. 3A. The ligand specificity of haSR-BI expressed in stably transfected ldlA cells was compared to that of the Var-261 cells, as shown in FIG. 3B. M-BSA inhibited the binding of ¹²⁵I-AcLDL to cells expressing haSR-BI and to Var-261 cells. Unexpectedly, haSR-BI was not inhibited by poly G, fucoidin or carrageenan (another classic scavenger receptor ligand (Brown and Goldstein, 1983)), which were competitors for the activity in Var-261 cells. In addition, LDL did not inhibit the binding of ¹²⁵I-AcLDL to Var-261 cells, but was a competitor for cells expressing haSR-BI. There is no other case in which native LDL has been reported to block scavenger receptor activity. Thus, the specificity of the haSR-BI differs dramatically from that reported for any previously described scavenger receptor, including that in Var-261 cells.

Northern blot analysis using haSR-BI as a probe showed that a single major mRNA (approximately 3.4 kb) was present in similar amounts in both the parental ldlA and Var-261 cells, indicating that the cells express similar amounts of haSR-BI mRNA. Therefore, although haSR-BI encodes a scavenger receptor, it is apparently expressed in both ldlA and Var-261 cells at very low levels. Taken together with the specificity data described above, these results indicate that haSR-BI is a novel scavenger receptor which was not responsible for most of the scavenger receptor activity in Var-261 cells.

Analysis of haSR-BI cDNA

The cloned haSR-BI cDNA is approximately 2.9 kb long. The sequences of the 5′ untranslated region, the coding region, and a portion of the 3′ untranslated region are shown in Sequence Listing ID No. 3. The predicted protein sequence is 509 amino acids (Sequence Listing ID No. 4) with a calculated molecular weight of 57 kD.

Based on comparison of amino acid sequence, haSR-BI has homology along its entire length to members of the CD36 family of membrane proteins: CD36 (32%, 31% and 33% amino acid identities with the human (Oquendo et al., 1989), murine (Endemann et al., 1993) and rat (also known as “FAT”, Abumrad et al., 1993) homologs), rat LIMPII (33%, a lysosomal integral membrane protein (Vega et al., 1991 J. Biol. Chem. 266, 16818-16824), and two Drosophila melanogaster proteins, emp (29%, Hart and Wilcox, 1993 J. Mol. Biol. 234, 249-253) and “dCD36” (31%, Genbank #DMCD361). All of these, with the exception of “dCD36”, have two internal hydrophobic domains, which have been suggested to serve as membrane spanning domains, and a series of conserved cysteines and putative N-linked glycosylation sites (see Vega et al., 1991 for a description of the common sequence elements).

Characterization and Comparison of haSR-BI Binding with Human CD36 Binding Affinity and Selectivity

To further characterize the binding properties of huCD36 and compare them with those of haSR-BI, the properties of COS cells transiently transfected with a cDNA expression vector for human CD36 (obtained from Dr. Brian Seed, Oquendo et al., 1989) were examined.

COS cells were transfected on day 1 with a plasmid encoding CD36. On day 2 the transfected cells were set in 6-well dishes at 1×10⁶ cells/well in medium A plus 1 mM sodium butyrate. On day 3, ¹²⁵I-AcLDL binding at 4° C. was measured. The high affinity values shown represent the differences between measurements made in the absence (duplicate incubations) and presence (single incubations) of an excess of the unlabeled competitor M-BSA (200 μg/ml). on day 3, degradation of ¹²⁵I-AcLDL (5 μl/ml) was measured after a 5 hr incubation at 37° C. in the absence (duplicate determinations) or presence (duplicate determinations) of the indicated competitors. The values represent the means of four determinations from two identical experiments (the error bars represent standard deviations). The 100% of control values (ng/5 hr/mg cell protein) from the two experiments were 232 and 103.

FIG. 4A shows that expression of huCD36 conferred high affinity ¹²⁵1I-AcLDL binding on transiently transfected COS cells at 4° C. with an apparent dissociation constant in the range of approximately 5 μg protein/ml. FIG. 4B shows that the receptor activity was inhibited by M-BSA and AcLDL, but not by other polyanions which inhibit class A macrophage scavenger receptors, including fucoidin, poly I, poly G, and ReLPS. FIG. 5 is a graph directly comparing lipoprotein inhibition of ¹²⁵I-AcLDL binding to haSR-BI and huCD36. The graph clearly demonstrates the similarities: i.e., binding to both receptors is inhibited by the modified LDL; and the differences: only haSR-BI binding is inhibited by LDL.

Subsequent competition binding studies demonstrate that HDL and VLDL (400 μg/ml) competitively inhibit binding of ¹²⁵I-AcLDL to haSR-BI, providing further support for the potential role of this receptor in lipoprotein and lipid metabolism.

Thus, the binding of AcLDL to huCD36 resembles the binding of AcLDL to haSR-BI, except that LDL effectively competes for ¹²⁵I-AcLDL binding to haSR-BI but not huCD36. These results suggest that haSR-BI and huCD36 define a new and distinct class of scavenger receptors which is designated class B scavenger receptors. Since the ligand specificities of the class B receptors are very different from the activity in Var-261 cells, it seems unlikely that either of these two class B scavenger receptors accounts for the binding activity observed in the Var-261 cells.

Tissue Distribution of haSR-BI

To explore the physiological functions of haSR-BI, the tissue distribution of haSR-BI was determined in murine tissues and during differentiation of 3T3-L1 cells into adipocytes using Northern blot. Each lane was loaded with 0.5 μg of poly(A)+ RNA prepared from the murine tissues: kidney, liver, brain, testis, fat, diaphragm, heart, lung, spleen, or from 3T3-L1 fibroblasts which were either nonconfluent at the fibroblast stage or confluent and induced to differentiate over a period of 0, 2, 4, 6 or 8 days. The blots were hybridized with a 600 base pair fragment of the coding region of haSR-BI. Hsp70 cDNA was used as a control for equal RNA loading. In the Northern of the murine tissues, the same blot was used for both SR-BI and hsp70 hybridizations. In the Northern of the differentiating 3T3-L1 cells, parallel blots were used. The blot of the murine tissues was also hybridized with a CD36 probe demonstrating that CD36 and SR-BI probes recognized different mRNA species.

One predominant band of approximately 2.4 kb was most abundant in fat and was present at moderate levels in lung and liver. There was little expression in the remaining tissues tested, which included kidney, brain, testis, diaphragm, heart, and spleen. To further investigate the expression of SR-BI in fat, the SR-BI mRNA levels in 3T3-L1 cells which were induced to differentiate into adipocytes were determined. The levels of SR-BI mRNA were found to increase during differentiation in a manner similar to that previously demonstrated for the glucose transporter GLUT4 and for Rab3D, a small molecular weight GTP binding protein (Baldini et al., 1992). These data, together with the data showing that modified LDL binding is inhibited by LDL, indicate that SR-BI plays a physiological role in lipid metabolism in adipocytes. In this regard, it is noteworthy that rat CD36 (also known as the “FAT” protein) was cloned as a result of its ability to directly bind reactive fatty acid esters (Abumrad et al., 1993). CD36 message is also markedly increased upon differentiation of the cultured lines 3T3 F442A and Ob1771 into adipocytes, as is the message for SR-BI in the 3T3-L1 adipocyte system. The expression patterns of SR-BI mRNA and CD36 mRNA are similar, but not identical. Both are found in high levels in adipose tissue. One notable difference in expression levels is found in the liver, where SR-BI expression is moderate but no CD36 message was detected. The observation that native LDL competes for AcLDL binding to SR-BI further indicated that SR-BI may play a role in lipid metabolism.

Even though CD36 and haSR-BI are structurally very different from the class A receptors, they apparently display similar complex binding properties (Freeman et al., 1991). However, the similarity does not extend to ligand specificity, since class B receptors are not inhibited by other nonproteinacious typical class A receptor polyanionic ligands, such as fucoidin and poly I. Thus, these class B scavenger receptors bind modified proteins, but not the broad range of polyanionic ligands which compose the distinct ligands of Var-261 cells and class A scavenger receptors.

Drosophila melanogaster Scavenger Receptor Protein

In an effort to further define the structures and functions of scavenger receptors, receptor expression was investigated in a representative invertebrate, Drosophila melanogaster (Abrams, et al. 1993 Proc. Natl. Acad. USA 89:10375-10379). By examining D. melanogaster embryos using fluorescently labeled AcLDL and ¹²⁵I-AcLDL as probes for receptor activity in vivo and in vitro, fluorescence was found to be distributed throughout the interstitial spaces of the body cavity in a pattern characteristic of the distribution of embryonic macrophages and was observed in cells with multivesicular inclusions characteristic of macrophages. Further analysis of primary embryonic cell cultures showed that uptake was macrophage specific and exhibited the broad specificity of mammalian scavenger receptors.

Two commonly used Drosophila cell lines were examined for scavenger receptor activity: L2 and Kc. Only the Schneider L2 cells, not the Kc cells, exhibit a scavenger receptor-mediated endocytic pathway, which is almost identical to that of mammalian macrophages. The L2 cell receptors exhibit characteristic scavenger receptor-like broad polyanion-binding specificity, and mediate high-affinity and saturable binding, uptake and degradation of AcLDL. In L2 cells, the kinetics of intracellular ligand degradation after binding and uptake shows a lag phase, intracellular ligand degradation is chloroquine sensitive, and endocytosis is temperature dependent.

Preparation of cDNA and Genomic Libraries

To facilitate both hybridization and expression cloning, cDNA libraries were generated from poly A+ RNA from L2 cells using standard procedures, as described above for Var-261 cells, and libraries were generated in the expression vector pcNDA1 (Invitrogen). The average insert size for the bidirectional L2 cell library is about 1.4 kb. DNAs for hybridization and transfection experiments were prepared as follows: E. coli strain MC1061/P3 is transformed with the cDNA expression library by electroporation and the transformed cells plated on LB-A/T plates (LB with Amp (15 μg/ml)/Tet (8 μg/ml)) to obtain isolated colonies at densities of 2,000 to 10,000 per 150 mm dish. Each plate, representing one pool, is scraped to recover the bacteria and a pool of library plasmids is isolated from each dish of bacteria using a midiprep method (miniprep method of Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., scaled up five to ten fold). The pools can be used for Southern blot analysis for hybridization screening and for transfection into COS or CHO cells for expression cloning.

Expression in Cultured Mammalian Cells

Expression of the receptors can be detected using either or both fluorescence microscopy and light microscopy of emulsion autoradiographs. Transient expression can be routinely obtained in COS cells, stable expression in CHO cells.

Fluorescence screening: Scavenger receptor activity can be detected using fluorescent, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate-labeled AcLDL (DiI-AcLDL). On day 0, COS M6 cells are plated in 35 mm culture dishes (3 to 4×10⁵ cells/dish) in medium A (DMEM supplemented with 100 units penicillin/ml medium, 100 μg streptomycin/ml medium, and 2 mM glutamine) containing 10% FBS (medium B). On day 1, the cells are transfected with 0.5 μg/dish of library DNA following the method of Cullen, 1987 Methods in Enz. 152:684-704. On day 2, monolayers were refed with modified medium B 1 mM sodium butyrate. On day 3, the monolayers are refed with modified medium B containing between one and five μg protein/ml of DiI-AcLDL. After a 5 hr incubation at 37° C., the plates are washed two times with PBS and the cells fixed with 3.7% formaldehyde in PBS for 10 min at room temperature. The presence of fluorescent DiI in the fixed cells is determined by visual screening using a Lietz inverted fluorescence microscope.

After screening the bidirectional L2 cell library, a pool of approximately 3500 clones which reproducibly conferred DiI-AcLDL endocytic activity in transiently transfected COS cells was identified. This pool was subdivided into 18 subpools of approximately 350 clones each, which were transfected into COS cells. One of these subpools also conferred DiI-AcLDL endocytic activity.

The clone from the subpool was isolated and transfected into CHO cells to yield CHO[dSR-CI]-2.6a cells. The nucleic acid (Sequence ID No. 5) and amino acid (Sequence ID No. 6) sequences were also obtained using the methods described above.

Binding Activity of CHO[dSR-CI]-2.6a Cells

Binding of ¹²⁵I-AcLDL to CHO[dSR-CI]-2.6a cells was measured as ng AcLDL protein bound/mg cell protein at concentrations of 1, 3, 6, 13, 25, and 50 μg AcLDL protein/ml. Uptake was also measured, comparing total, nonspecific and specific binding.

As shown by FIG. 6A, dSR-CI binds AcLDL. As shown by FIG. 6B, AcLDL was also taken up by the cells expressing dSR-CI.

Degradation was measured to confirm that dSR-CI mediated internalization of the AcLDL. The results shown in FIG. 6C confirm the selective degradation of AcLDL.

Competitive binding of AcLDL was performed to more accurately define the specificity of the AcLDL binding. The results are shown below in Table 1.

TABLE 1 Competitive Inhibition of ¹²⁵I-AcLDL Degradation Activity by CHO[dSR-CI]-2.6a Cells. Competitor Degradation (% No Competitor) None 100 ACLDL (400 μg/ml) 4.8 LDL (400 μg/ml) 97.6 M-BSA (400 μg/ml) 6.7 BSA (400 μg/ml) 94.4 poly I (400 μg/ml) 2.7 dA5G37 (100 μg/ml) 5.1 dA37 (100 μg/ml) 98.2 dA37 (400 μg/ml) 43.0 Dextran Sulfate 4.2 Dextran (400 μg/ml) 100 Poly D-glutamic acid 34 (400 μg/ml)

Applications of the Scavenger Receptor Proteins

The presence of scavenger receptors on both mammalian and Drosophila macrophages suggests that they mediate critical, well-conserved functions, possibly pathogen recognition, and raises the possibility that they may have appeared early in the evolution of host defense systems. In this regard, it is known that postembryonic macrophagelike hemocytes in Drosophila participate in wound healing, encapsulation of pathogens, and phagocytosis. Furthermore, macrophages play an important role in the recognition of apoptotic or senescent cells during the course of development, normal cell turnover, and aging, although it is not known if the scavenger receptors are also involved in these processes.

Accordingly, the understanding of the structure and functions of the receptor proteins described herein, as well as the cDNAs encoding these proteins, have a variety of uses. Specifically, the proteins and their DNAs can be used in screening of drugs which modulate the activity and/or the expression of the receptors; in screening of patient samples for the presence of functional receptor protein; in the case of the SR-BI receptor protein, removal of LDL and modified LDL by reaction with immobilized receptor protein; use of the DNA to construct probes for screening of libraries for other receptors, including the human equivalents, and the regulatory sequences controlling the expression of the other receptors as well as SR-BI and SR-CI. These drugs, when identified, may be useful in treating or preventing atherosclerosis, fat uptake by adipocytes, and some types of immune disorders.

Isolation of Other Receptor Proteins

The nucleotide sequences identified herein as encoding hamster SR-BI and Drosophila melanogaster SR-CI are useful as probes for screening of libraries for the presence of related receptors. Libraries are constructed from cells of a desired species, such as humans, which are then screened with all or a portion of the nucleotide sequence encoding either SR-BI or SR-CI. Specific regions of interest are those portions of the nucleotide sequence which encode regions of the protein conserved between different receptors; between the same receptors from different species; and within discrete regions of the receptor proteins: the cytoplasmic region, the transmembrane region, the “stem” regions that may include EGF repeats, collagen like regions α-helical coiled regions, or regions having a high density of cysteines (CCP domains), and specific ligand regions. These regions are identified by structural analysis such as that which has been used to generate the schematics in FIGS. 1A and 1B, using methods routinely available to those skilled in the art. These methods include chemical crosslinking, electrophoretic analysis, hydrodynamic studies, and electron microscopy and computer assisted analysis of structure based on predicted amino acid sequence.

As used herein, unless specifically stated otherwise, the term “SR-BI” refers to the sequences shown in Sequence ID Nos. 3 and 4, and degenerate variants thereof and their equivalents in other species of origin, especially human, as well as functionally equivalent variants, having additions, deletions, and substitutions of either nucleotides or amino acids which do not significantly alter the functional activity of the protein as a receptor characterized by the binding activity identified above. The term “SR-CI” refers to the sequences shown in Sequence ID Nos. 5 and 6, and degenerate variants thereof and their equivalents in other species of origin, especially human, as well as functionally equivalent variants, having additions, deletions, and substitutions of either nucleotides or amino acids which do not significantly alter the functional activity of the protein as a receptor characterized by the binding activity identified above.

Preferred uses for these sequences, especially those in the Sequence Listings below, are for the cloning of equivalent receptor molecules present in human cells, for the isolation and characterization of the regulatory sequences present in the genome which controlled the extent to which a particular receptor is expressed in a cell, and for the screening of drugs altering binding of or endocytosis of ligand by the scavenger receptor proteins.

Isolation of Receptor Proteins

Additional receptor proteins for study can be obtained by expression in suitable recombinant host systems, such as mammalian, yeast, bacteria, or insect cells. Isolation can be facilitated by making antibodies to the recombinant protein which are then immobilized on substrates for use in purification of additional receptors.

Screening of Patient Samples for Expression of Receptor Proteins

The sequences disclosed herein are useful in screening of patient samples for the presence of normal receptor proteins, using hybridization assays of patient samples, including blood and tissues. Screening can also be accomplished using antibodies, typically labelled with a fluorescent, radiolabelled, or enzymatic label, or by isolation of target cells and screening for binding activity, as described in the examples above. Typically, one would be screening for expression on either a qualitative or quantitative basis, and for expression of functional receptor.

Hybridization Probes

Reaction conditions for hybridization of an oligonucleotide probe or primer to a nucleic acid sequence vary from oligonucleotide to oligonucleotide, depending on factors such as oligonucleotide length, the number of G and C nucleotides, and the composition of the buffer utilized in the hybridization reaction. Moderately stringent hybridization conditions are generally understood by those skilled in the art as conditions approximately 25° C. below the melting temperature of a perfectly base-paired double-stranded DNA. Higher specificity is generally achieved by employing incubation conditions having higher temperatures, in other words more stringent conditions. In general, the longer the sequence or higher the G and C content, the higher the temperature and/or salt the lower concentration required. Chapter 11 of the well-known laboratory manual of Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, second edition, Cold Spring Harbor Laboratory Press, New York (1990) (which is incorporated by reference herein), describes hybridization conditions for oligonucleotide probes and primers in great detail, including a description of the factors involved and the level of stringency necessary to guarantee hybridization with specificity.

The preferred size of a hybridization probe is from 10 nucleotides to 100,000 nucleotides in length. Below 10 nucleotides, hybridized systems are not stable and will begin to denature above 20° C. Above 100,000 nucleotides, one finds that hybridization (renaturation) becomes a much slower and incomplete process, as described in greater detail in the text MOLECULAR GENETICS, Stent, G. S. and R. Calender, pp. 213-219 (1971). Ideally, the probe should be from 20 to 10,000 nucleotides. Smaller nucleotide sequences (20-100) lend themselves to production by automated organic synthetic techniques. Sequences from 100-10,000 nucleotides can be obtained from appropriate restriction endonuclease treatments. The labeling of the smaller probes with the relatively bulky chemiluminescent moieties may in some cases interfere with the hybridization process.

Generation of Antibodies for Diagnostic or Therapeutic Use

Antibodies to the receptor proteins can also be generated which are useful in detection, characterization or isolation of receptor proteins, as well as for modifying receptor protein activity, in most cases, through inhibition of binding. Antibodies are generated by standard techniques, using human or animal receptor proteins. Since the proteins exhibit high evolutionary conservation, it may be advantageous to generate antibodies to a protein of a different species of origin than the species in which the antibodies are to be tested or utilized, looking for those antibodies which are immunoreactive with the most evolutionarily conserved regions. Antibodies are typically generated by immunization of an animal using an adjuvant such as Freund's adjuvant in combination with an immunogenic amount of the protein administered over a period of weeks in two to three week intervals, then isolated from the serum, or used to make hybridomas which express the antibodies in culture. Because the methods for immunizing animals yield antibody which is not of human origin, the antibodies could elicit an adverse effect if administered to humans. Methods for “humanizing” antibodies, or generating less immunogenic fragments of non-human antibodies, are well known. A humanized antibody is one in which only the antigen-recognized sites, or complementarily-determining hypervariable regions (CDRs) are of non-human origin, whereas all framework regions (FR) of variable domains are products of human genes. These “humanized” antibodies present a lesser xenographic rejection stimulus when introduced to a human recipient.

To accomplish humanization of a selected mouse monoclonal antibody, the CDR grafting method described by Daugherty, et al., 1991 Nucl. Acids Res., 19:2471-2476, incorporated herein by reference, may be used. Briefly, the variable region DNA of a selected animal recombinant anti-idiotypic ScFv is sequenced by the method of Clackson, T., et al., 1991 Nature, 352:624-688, incorporated herein by reference. Using this sequence, animal CDRs are distinguished from animal framework regions (FR) based on locations of the CDRs in known sequences of animal variable genes. Kabat, H. A., et al., Sequences of Proteins of Immunological Interest, 4th Ed. (U.S. Dept. Health and Human Services, Bethesda, Md., 1987). Once the animal CDRs and FR are identified, the CDRs are grafted onto human heavy chain variable region framework by the use of synthetic oligonucleotides and polymerase chain reaction (PCR) recombination. Codons for the animal heavy chain CDRs, as well as the available human heavy chain variable region framework, are built in four (each 100 bases long) oligonucleotides. Using PCR, a grafted DNA sequence of 400 bases is formed that encodes for the recombinant animal CDR/human heavy chain FR protection.

The immunogenic stimulus presented by the monoclonal antibodies so produced may be further decreased by the use of Pharmacia's (Pharmacia LKB Biotechnology, Sweden) “Recombinant Phage Antibody System” (RPAS), which generates a single-chain Fv fragment (ScFv) which incorporates the complete antigen-binding domain of the antibody. In the RPAS, antibody variable heavy and light chain genes are separately amplified from the hybridoma mRNA and cloned into an expression vector. The heavy and light chain domains are co-expressed on the same polypeptide chain after joining with a short linker DNA which codes for a flexible peptide. This assembly generates a single-chain Fv fragment (ScFv) which incorporates the complete antigen-binding domain of the antibody. Compared to the intact monoclonal antibody, the recombinant ScFv includes a considerably lower number of epitopes, and thereby presents a much weaker immunogenic stimulus when injected into humans.

The antibodies can be formulated in standard pharmaceutical carriers for administration to patients in need thereof. These include saline, phosphate buffered saline, and other aqueous carriers, and liposomes, polymeric microspheres and other controlled release delivery devices, as are well known in the art. The antibodies can also be administered with adjuvant, such as muramyl dipeptide or other materials approved for use in humans (Freund's adjuvant can be used for administration of antibody to animals).

Screening for Drugs Modifying or Altering the Extent of Receptor Function or Expression

The receptor proteins are useful as targets for compounds which turn on, or off, or otherwise regulate binding to these receptors. The assays described above clearly provide routine methodology by which a compound can be tested for an inhibitory effect on binding of a specific compound, such as a radiolabelled modified LDL or polyion. The in vitro studies of compounds which appear to inhibit binding selectively to the receptors are then confirmed by animal testing. Since the molecules are so highly evolutionarily conserved, it is possible to conduct studies in laboratory animals such as mice to predict the effects in humans.

Studies based on inhibition of binding are predictive for indirect effects of alteration of receptor binding. For example, inhibition of LDL binding to the SR-BI receptor leads to decreased uptake by cells of LDL and therefore decreases deposition of LDL in cells; similar effects should be observed for inhibition of lipoprotein and/or lipid by adipocytes expressing the SR-BI receptor. Conversely, increasing LDL binding to cells increases removal of lipids from the blood stream and thereby decreases lipid deposition within the blood stream. Studies have been conducted using a stimulator to enhance macrophage uptake of cholesterol and thereby treat atherogenesis, using M-CSF (Schaub, et al., 1994 Arterioscler. Thromb. 14(1), 70-76; Inaba, et al., 1993 J. Clin. Invest. 92(2), 750-757). Although the target of the stimulator is not known with specificity, this provides further support for the rationale for believing the indirect in vivo effects can be achieved based on the in vitro binding data.

Assays for testing compounds for useful activity can be based solely on interaction with the receptor protein, preferably expressed on the surface of transfected cells such as those described above, although proteins in solution or immobilized on inert substrates can also be utilized, where the indication is inhibition or increase in binding of LDL or modified LDL.

Alternatively, the assays can be based on interaction with the gene sequence encoding the receptor protein, preferably the regulatory sequences directing expression of the receptor protein. For example, antisense which binds to the regulatory sequences, and/or to the protein encoding sequences can be synthesized using standard oligonucleotide synthetic chemistry. The antisense can be stabilized for pharmaceutical use using standard methodology (encapsulation in a liposome or microsphere; introduction of modified nucleotides that are resistant to degradation or groups which increase resistance to endonucleases, such as phosphorothiodates and methylation), then screened initially for alteration of receptor activity in transfected or naturally occurring cells which express the receptor, then in vivo in laboratory animals. Typically, the antisense would inhibit expression. However, sequences which block those sequences which “turn off” synthesis can also be targeted.

The receptor protein for study can be isolated from either naturally occurring cells or cells which have been genetically engineered to express the receptor, as described in the examples above. In the preferred embodiment, the cells would have been engineered using the intact gene.

Random Generation of Receptor or Receptor Encoding Sequence Binding Molecules

Molecules with a given function, catalytic or ligand-binding, can be selected for from a complex mixture of random molecules in what has been referred to as “in vitro genetics” (Szostak, TIBS 19:89, 1992). One synthesizes a large pool of molecules bearing random and defined sequences and subjects that complex mixture, for example, approximately 10¹⁵ individual sequences in 100 μg of a 100 nucleotide RNA, to some selection and enrichment process. For example, by repeated cycles of affinity chromatography and PCR amplification of the molecules bound to the ligand on the column, Ellington and Szostak (1990) estimated that 1 in 10¹⁰ RNA molecules folded in such a way as to bind a given ligand. DNA molecules with such ligand-binding behavior have been isolated (Ellington and Szostak, 1992; Bock et al, 1992).

Computer Assisted Drug Design

Computer modeling technology allows visualization of the three-dimensional atomic structure of a selected molecule and the rational design of new compounds that will interact with the molecule. The three-dimensional construct typically depends on data from x-ray crystallographic analyses or NMR imaging of the selected molecule. The molecular dynamics require force field data. The computer graphics systems enable prediction of how a new compound will link to the target molecule and allow experimental manipulation of the structures of the compound and target molecule to perfect binding specificity. Prediction of what the molecule-compound interaction will be when small changes are made in one or both requires molecular mechanics software and computationally intensive computers, usually coupled with user-friendly, menu-driven interfaces between the molecular design program and the user.

Examples of molecular modelling systems are the CHARMm and QUANTA programs, Polygen Corporation, Waltham, Mass. CHARMm performs the energy minimization and molecular dynamics functions. QUANTA performs the construction, graphic modelling and analysis of molecular structure. QUANTA allows interactive construction, modification, visualization, and analysis of the behavior of molecules with each other.

A number of articles review computer modeling of drugs interactive with specific proteins, such as Rotivinen, et al., 1988 Acta Pharmaceutica Fennica 97, 159-166; Ripka, New Scientist 54-57 (Jun. 16, 1988); McKinaly and Rossmann, 1989 Annu. Rev. Pharmacol. Toxiciol. 29, 111-122; Perry and Davies, QSAR: Quantitative Structure-Activity Relationships in Drug Design pp. 189-193 (Alan R. Liss, Inc. 1989); Lewis and Dean, 1989 Proc. R. Soc. Lond. 236, 125-140 and 141-162; and, with respect to a model receptor for nucleic acid components, Askew, et al., 1989 J. Am. Chem. Soc. 111, 1082-1090. Other computer programs that screen and graphically depict chemicals are available from companies such as BioDesign, Inc., Pasadena, Calif., Allelix, Inc, Mississauga, Ontario, Canada, and Hypercube, Inc., Cambridge, Ontario. Although these are primarily designed for application to drugs specific to particular proteins, they can be adapted to design of drugs specific to regions of DNA or RNA, once that region is identified.

Although described above with reference to design and generation of compounds which could alter binding, one could also screen libraries of known compounds, including natural products or synthetic chemicals, and biologically active materials, including proteins, for compounds which are inhibitors or activators.

Generation of Nucleic Acid Regulators

Nucleic acid molecules containing the 5′ regulatory sequences of the receptor genes can be used to regulate or inhibit gene expression in vivo. Vectors, including both plasmid and eukaryotic viral vectors, may be used to express a particular recombinant 5′ flanking region-gene construct in cells depending on the preference and judgment of the skilled practitioner (see, e.g., Sambrook et al., Chapter 16). Furthermore, a number of viral and nonviral vectors are being developed that enable the introduction of nucleic acid sequences in vivo (see, e.g., Mulligan, 1993 Science, 260, 926-932; U.S. Pat. Nos. 4,980,286; 4,868,116; incorporated herein by reference). Recently, a delivery system was developed in which nucleic acid is encapsulated in cationic liposomes which can be injected intravenously into a mammal. This system has been used to introduce DNA into the cells of multiple tissues of adult mice, including endothelium and bone marrow (see, e.g., Zhu et al., 1993 Science 261, 209-211; incorporated herein by reference).

The 5′ flanking sequences of the receptor gene can also be used to inhibit the expression of the receptor. For example, an antisense RNA of all or a portion of the 5′ flanking region of the receptor gene can be used to inhibit expression of the receptor in vivo. Expression vectors (e.g., retroviral expression vectors) are already available in the art which can be used to generate an antisense RNA of a selected DNA sequence which is expressed in a cell (see, e.g., U.S. Pat. Nos. 4,868,116; 4,980,286). Accordingly, DNA containing all or a portion of the sequence of the 5′ flanking region of the receptor gene can be inserted into an appropriate expression vector so that upon passage into the cell, the transcription of the inserted DNA yields an antisense RNA that is complementary to the mRNA transcript of the receptor protein gene normally found in the cell. This antisense RNA transcript of the inserted DNA can then base-pair with the normal mRNA transcript found in the cell and thereby prevent the mRNA from being translated. It is of course necessary to select sequences of the 5′ flanking region that are downstream from the transcriptional start sites for the receptor protein gene to ensure that the antisense RNA contains complementary sequences present on the mRNA. Antisense RNA can be generated in-vitro also, and then inserted into cells. Oligonucleotides can be synthesized on an automated synthesizer (e.g., Model 8700 automated synthesizer of Milligen-Biosearch, Burlington, Mass. or ABI Model 380B). In addition, antisense deoxyoligonucleotides have been shown to be effective in inhibiting gene transcription and viral replication (see e.g., Zamecnik et al., 1978 Proc. Natl. Acad. Sci. USA 75, 280-284; Zamecnik et al., 1986 Proc. Natl. Acad. Sci., 83, 4143-4146; Wickstrom et al., 1988 Proc. Natl. Acad. Sci. USA 85, 1028-1032; Crooke, 1993 FASEB J. 7, 533-539. Furthermore, recent work has shown that improved inhibition of expression of a gene by antisense oligonucleotides is possible if the antisense oligonucleotides contain modified nucleotides (see, e.g., Offensperger et. al., 1993 EMBO J. 12, 1257-1262 (in vivo inhibition of duck hepatitis B viral replication and gene expression by antisense phosphorothioate oligodeoxynucleotides); Rosenberg et al., PCT WO 93/01286 (synthesis of sulfurthioate oligonucleotides); Agrawal et al., 1988 Proc. Natl. Acad. Sci. USA 85, 7079-7083 (synthesis of antisense oligonucleoside phosphoramidates and phosphorothioates to inhibit replication of human immunodeficiency virus-l); Sarin et al., 1989 Proc. Natl. Acad. Sci. USA 85, 7448-7794 (synthesis of antisense methylphosphonate oligonucleotides); Shaw et al., 1991 Nucleic Acids Res 19, 747-750 (synthesis of 3′ exonuclease-resistant oligonucleotides containing 3′ terminal phosphoroamidate modifications); incorporated herein by reference).

The sequences of the 5′ flanking region of receptor protein gene can also be used in triple helix (triplex) gene therapy. Oligonucleotides complementary to gene promoter sequences on one of the strands of the DNA have been shown to bind promoter and regulatory sequences to form local triple nucleic acid helices which block transcription of the gene (see, e.g., 1989 Maher et al., Science 245, 725-730; Orson et al., 1991 Nucl. Acids Res. 19, 3435-3441; Postal et al., 1991 Proc. Natl. Acad. Sci. USA 88, 8227-8231; Cooney et al., 1988 Science 241, 456-459; Young et al., 1991 Proc. Natl. Acad. Sci. USA 88, 10023-10026; Duval-Valentin et al., 1992 Proc. Natl. Acad. Sci. USA 89, 504-508; 1992 Blume et al., Nucl. Acids Res. 20, 1777-1784; 1992 Grigoriev et al., J. Biol. Chem. 267, 3389-3395.

Recently, both theoretical calculations and empirical findings have been reported which provide guidance for the design of oligonucleotides for use in oligonucleotide-directed triple helix formation to inhibit gene expression. For example, oligonucleotides should generally be greater than 14 nucleotides in length to ensure target sequence specificity (see, e.g., Maher et al., (1989); Grigoriev et al., (1992)). Also, many cells avidly take up oligonucleotides that are less than 50 nucleotides in length (see e.g., Orson et al., (1991); Holt et al., 1988 Mol. Cell. Biol. 8, 963-973; Wickstrom et al., 1988 Proc. Natl. Acad. Sci. USA 85, 1028-1032). To reduce susceptibility to intracellular degradation, for example by 3′ exonucleases, a free amine can be introduced to a 3′ terminal hydroxyl group of oligonucleotides without loss of sequence binding specificity (Orson et al., 1991). Furthermore, more stable triplexes are formed if any cytosines that may be present in the oligonucleotide are methylated, and also if an intercalating agent, such as an acridine derivative, is covalently attached to a 5′ terminal phosphate (e.g., via a pentamethylene bridge); again without loss of sequence specificity (Maher et al., (1989); Grigoriev et al., (1992).

Methods to produce or synthesize oligonucleotides are well known in the art. Such methods can range from standard enzymatic digestion followed by nucleotide fragment isolation (see e.g., Sambrook et al., Chapters 5, 6) to purely synthetic methods, for example, by the cyanoethyl phosphoramidite method using a Milligen or Beckman System 1Plus DNA synthesizer (see also, Ikuta et al., in Ann. Rev. Biochem. 1984 53, 323-356 (phosphotriester and phosphite-triester methods); Narang et al., in Methods Enzymol., 65, 610-620 (1980) (phosphotriester method). Accordingly, DNA sequences of the 5′ flanking region of the receptor protein gene described herein can be used to design and construct oligonucleotides including a DNA sequence consisting essentially of at least 15 consecutive nucleotides, with or without base modifications or intercalating agent derivatives, for use in forming triple helices specifically within the 5′ flanking region of a receptor protein gene in order to inhibit expression of the gene.

In some cases it may be advantageous to insert enhancers or multiple copies of the regulatory sequences into an expression system to facilitate screening of methods and reagents for manipulation of expression.

Preparation of Receptor Protein Fragments

Compounds which are effective for blocking binding of the receptor can also consist of fragments of the receptor proteins, expressed recombinantly and cleaved by enzymatic digest or expressed from a sequence encoding a peptide of less than the full length receptor protein. These will typically be soluble proteins, i.e., not including the transmembrane and cytoplasmic regions, although smaller portions determined in the assays described above to inhibit or compete for binding to the receptor proteins can also be utilized. It is a routine matter to make appropriate receptor protein fragments, test for binding, and then utilize. The preferred fragments are of human origin, in order to minimize potential immunological response. The peptides can be as short as five to eight amino acids in length and are easily prepared by standard techniques. They can also be modified to increase in vivo half-life, by chemical modification of the amino acids or by attachment to a carrier molecule or inert substrate. Based on studies with other peptide fragments blocking receptor binding, the IC₅₀, the dose of peptide required to inhibit binding by 50%, ranges from about 50 μM to about 300 μM, depending on the peptides. These ranges are well within the effective concentrations for the in vivo administration of peptides, based on comparison with the RGD-containing peptides, described, for example, in U.S. Pat. No. 4,792,525 to Ruoslaghti, et al., used in vivo to alter cell attachment and phagocytosis. The peptides can also be conjugated to a carrier protein such as keyhole limpet hemocyanin by its N-terminal cysteine by standard procedures such as the commercial Imject kit from Pierce Chemicals or expressed as a fusion protein, which may have increased efficacy. As noted above, the peptides can be prepared by proteolytic cleavage of the receptor proteins, or, preferably, by synthetic means. These methods are known to those skilled in the art. An example is the solid phase synthesis described by J. Merrifield, 1964 J. Am. Chem. Soc. 85, 2149, used in U.S. Pat. No. 4,792,525, and described in U.S. Pat. No. 4,244,946, wherein a protected alpha-amino acid is coupled to a suitable resin, to initiate synthesis of a peptide starting from the C-terminus of the peptide. Other methods of synthesis are described in U.S. Pat. Nos. 4,305,872 and 4,316,891. These methods can be used to synthesize peptides having identical sequence to the receptor proteins described herein, or substitutions or additions of amino acids, which can be screened for activity as described above.

The peptide can also be administered as a pharmaceutically acceptable acid- or base-addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.

Peptides containing cyclopropyl amino acids, or amino acids derivatized in a similar fashion, can also be used. These peptides retain their original activity but have increased half-lives in vivo. Methods known for modifying amino acids, and their use, are known to those skilled in the art, for example, as described in U.S. Pat. No. 4,629,784 to Stammer.

The peptides are generally active when administered parenterally in amounts above about 1 μg/kg of body weight. Based on extrapolation from other proteins, for treatment of most inflammatory disorders, the dosage range will be between 0.1 to 70 mg/kg of body weight. This dosage will be dependent, in part, on whether one or more peptides are administered.

Pharmaceutical Compositions

Compounds which alter receptor protein binding are preferably administered in a pharmaceutically acceptable vehicle. Suitable pharmaceutical vehicles are known to those skilled in the art. For parenteral administration, the compound will usually be dissolved or suspended in sterile water or saline. For enteral administration, the compound will be incorporated into an inert carrier in tablet, liquid, or capsular form. Suitable carriers may be starches or sugars and include lubricants, flavorings, binders, and other materials of the same nature. The compounds can also be administered locally by topical application of a solution, cream, gel, or polymeric material (for example, a Pluronic™, BASF).

Alternatively, the compound may be administered in liposomes or microspheres (or microparticles). Methods for preparing liposomes and microspheres for administration to a patient are known to those skilled in the art. U.S. Pat. No. 4,789,734 describe methods for encapsulating biological materials in liposomes. Essentially, the material is dissolved in an aqueous solution, the appropriate phospholipids and lipids added, along with surfactants if required, and the material dialyzed or sonicated, as necessary. A review of known methods is by G. Gregoriadis, Chapter 14. “Liposomes”, Drug Carriers in Biology and Medicine pp. 287-341 (Academic Press, 1979). Microspheres formed of polymers or proteins are well known to those skilled in the art, and can be tailored for passage through the gastrointestinal tract directly into the bloodstream. Alternatively, the compound can be incorporated and the microspheres, or composite of microspheres, implanted for slow release over a period of time, ranging from days to months. See, for example, U.S. Pat. Nos. 4,906,474, 4,925,673, and 3,625,214.

Removal of LDL from Patients or Patient Samples

The SR-BI receptor proteins can be used to remove LDL from patient blood, by immobilizing the receptor on a suitable substrate, such as the cellulose membrane of a dialysis unit, using conventional coupling, for example, using carboimide. The patient's blood is then dialyzed through the unit.

Modifications and variations of the methods and materials described herein will be obvious to those skilled in the art and are intended to be encompassed by the following claims. The teachings of the references cited herein are specifically incorporated herein.

6 20 base pairs nucleic acid single linear DNA NO NO et al.Ashkenas J. Lipid Res. 34 983-1000 1993 1 FROM 1 TO 20 1 AATGAAGAAC TGCTTAGTTT 20 18 base pairs nucleic acid single linear DNA NO NO et al.Ashkenas J. Lipid Res. 34 983-1000 1993 1 FROM 1 TO 18 2 AATCAAGGAA TTTAACTG 18 1788 base pairs nucleic acid double linear cDNA NO NO misc_feature 156..1683 /function= “Nucleotides 156 through 1683 encode the amino 3 GCCACCTGCA GGGCTACTGC TGCTCCGGCC ACTGCCTGAG ACTCACCTTG CTGGAACGTG 60 AGCCTCGGCT TCTGTCATCT CTGTGGCCTC TGTCGCTTCT GTCGCTGTCC CCCTTCAGTC 120 CCTGAGCCCC GCGAGCCCGG GCCGCACACG CGGACATGGG CGGCAGCGCC AGGGCGCGCT 180 GGGTGGCGGT GGGGCTGGGC GTCGTGGGGC TGCTGTGCGC TGTGCTCGGT GTGGTTATGA 240 TCCTCGTGAT GCCCTCGCTC ATCAAACAGC AGGTACTGAA GAATGTCCGC ATAGACCCCA 300 GCAGCCTGTC CTTTGCAATG TGGAAGGAGA TCCCTGTACC CTTCTACTTG TCCGTCTACT 360 TCTTCGAGGT GGTCAATCCC AGCGAGATCC TAAAGGGTGA GAAGCCAGTA GTGCGGGAGC 420 GTGGACCCTA TGTCTACAGG GAATTCAGAC ATAAGGCCAA CATCACCTTC AATGACAATG 480 ATACTGTGTC CTTTGTGGAG CACCGCAGCC TCCATTTCCA GCCGGACAGG TCCCACGGCT 540 CTGAGAGTGA CTACATTATA CTGCCTAACA TTCTGGTCTT GGGGGGCGCA GTAATGATGG 600 AGAGCAAGTC TGCAGGCCTG AAGCTGATGA TGACCTTGGG GCTGGCCACC TTGGGCCAGC 660 GTGCCTTTAT GAACCGAACA GTTGGTGAGA TCCTGTGGGG CTATGAGGAT CCCTTCGTGA 720 ATTTTATCAA CAAATACTTA CCAGACATGT TCCCCATCAA GGGCAAGTTC GGCCTGTTTG 780 TTGAGATGAA CAACTCAGAC TCTGGGCTCT TCACTGTGTT CACGGGCGTC CAGAACTTCA 840 GCAAGATCCA CCTGGTGGAC AGATGGAATG GGCTCAGCAA GGTCAACTAC TGGCATTCAG 900 AGCAGTGCAA CATGATCAAT GGCACTTCCG GGCAGATGTG GGCACCATTC ATGACACCCC 960 AGTCCTCGCT GGAATTCTTC AGTCCGGAAG CCTGCAGGTC TATGAAGCTC ACCTACCATG 1020 ATTCAGGGGT GTTTGAAGGC ATCCCCACCT ATCGCTTCAC AGCCCCTAAA ACTTTGTTTG 1080 CCAATGGGTC TGTTTACCCA CCCAATGAAG GTTTCTGCCC GTGCCTTGAA TCCGGCATTC 1140 AAAATGTCAG CACTTGCAGG TTTGGTGCAC CCCTGTTTCT GTCACACCCT CACTTCTACA 1200 ATGCAGACCC TGTGCTATCA GAAGCCGTTC TGGGTCTGAA CCCTGACCCA AGGGAGCATT 1260 CTTTGTTCCT TGACATCCAT CCGGTCACTG GGATCCCCAT GAACTGTTCT GTGAAGTTGC 1320 AGATAAGCCT CTACATCAAA GCTGTCAAGG GCATTGGGCA AACAGGGAAG ATCGAGCCCG 1380 TGGTCCTCCC ATTGCTGTGG TTTGAGCAGA GCGGTGCCAT GGGCGGCGAG CCCCTGAACA 1440 CGTTCTACAC GCAGCTGGTG CTGATGCCCC AGGTACTTCA GTATGTGCAG TATGTGCTGC 1500 TGGGGCTGGG CGGCCTCCTG CTGCTGGTGC CCGTCATCTA CCAGTTGCGC AGCCAGGAGA 1560 AATGCTTTTT ATTTTGGAGT GGTAGTAAAA AGGGCTCGCA GGATAAGGAG GCCATTCAGG 1620 CCTACTCTGA GTCTCTGATG TCACCAGCTG CCAAGGGCAC GGTGCTGCAA GAAGCCAAGC 1680 TGTAGGGTCC CAAAGACACC ACGAGCCCCC CCAACCTGAT AGCTTGGTCA GACCAGCCAT 1740 CCAGCCCCTA CACCCCGCTT CTTGAGGACT CTCTCAGCGG ACAGTCGC 1788 509 amino acids amino acid linear protein NO internal misc_feature 1..509 /function= ”Amino acid sequence for the Hamster Scavenger Domain 9..32 /note= “Putative transmembrane domain.” Domain 440..464 /note= “Putative transmembrane domain.” Modified-site 1..385 /note= “Positions 102-104, 108-110, 173-175, 212-214, 227-229, 255-257,310-312, 330-332 and 383-385 represent potential N-linked glycosylation sites.” Modified-site 21..470 /note= “The cysteines at positions 21, 251, 280, 321, 323, 334, 384 and 470 represent potential disulfide linkages.” 4 Met Gly Gly Ser Ala Arg Ala Arg Trp Val Ala Val Gly Leu Gly Val 1 5 10 15 Val Gly Leu Leu Cys Ala Val Leu Gly Val Val Met Ile Leu Val Met 20 25 30 Pro Ser Leu Ile Lys Gln Gln Val Leu Lys Asn Val Arg Ile Asp Pro 35 40 45 Ser Ser Leu Ser Phe Ala Met Trp Lys Glu Ile Pro Val Pro Phe Tyr 50 55 60 Leu Ser Val Tyr Phe Phe Glu Val Val Asn Pro Ser Glu Ile Leu Lys 65 70 75 80 Gly Glu Lys Pro Val Val Arg Glu Arg Gly Pro Tyr Val Tyr Arg Glu 85 90 95 Phe Arg His Lys Ala Asn Ile Thr Phe Asn Asp Asn Asp Thr Val Ser 100 105 110 Phe Val Glu His Arg Ser Leu His Phe Gln Pro Asp Arg Ser His Gly 115 120 125 Ser Glu Ser Asp Tyr Ile Ile Leu Pro Asn Ile Leu Val Leu Gly Gly 130 135 140 Ala Val Met Met Glu Ser Lys Ser Ala Gly Leu Lys Leu Met Met Thr 145 150 155 160 Leu Gly Leu Ala Thr Leu Gly Gln Arg Ala Phe Met Asn Arg Thr Val 165 170 175 Gly Glu Ile Leu Trp Gly Tyr Glu Asp Pro Phe Val Asn Phe Ile Asn 180 185 190 Lys Tyr Leu Pro Asp Met Phe Pro Ile Lys Gly Lys Phe Gly Leu Phe 195 200 205 Val Glu Met Asn Asn Ser Asp Ser Gly Leu Phe Thr Val Phe Thr Gly 210 215 220 Val Gln Asn Phe Ser Lys Ile His Leu Val Asp Arg Trp Asn Gly Leu 225 230 235 240 Ser Lys Val Asn Tyr Trp His Ser Glu Gln Cys Asn Met Ile Asn Gly 245 250 255 Thr Ser Gly Gln Met Trp Ala Pro Phe Met Thr Pro Gln Ser Ser Leu 260 265 270 Glu Phe Phe Ser Pro Glu Ala Cys Arg Ser Met Lys Leu Thr Tyr His 275 280 285 Asp Ser Gly Val Phe Glu Gly Ile Pro Thr Tyr Arg Phe Thr Ala Pro 290 295 300 Lys Thr Leu Phe Ala Asn Gly Ser Val Tyr Pro Pro Asn Glu Gly Phe 305 310 315 320 Cys Pro Cys Leu Glu Ser Gly Ile Gln Asn Val Ser Thr Cys Arg Phe 325 330 335 Gly Ala Pro Leu Phe Leu Ser His Pro His Phe Tyr Asn Ala Asp Pro 340 345 350 Val Leu Ser Glu Ala Val Leu Gly Leu Asn Pro Asp Pro Arg Glu His 355 360 365 Ser Leu Phe Leu Asp Ile His Pro Val Thr Gly Ile Pro Met Asn Cys 370 375 380 Ser Val Lys Leu Gln Ile Ser Leu Tyr Ile Lys Ala Val Lys Gly Ile 385 390 395 400 Gly Gln Thr Gly Lys Ile Glu Pro Val Val Leu Pro Leu Leu Trp Phe 405 410 415 Glu Gln Ser Gly Ala Met Gly Gly Glu Pro Leu Asn Thr Phe Tyr Thr 420 425 430 Gln Leu Val Leu Met Pro Gln Val Leu Gln Tyr Val Gln Tyr Val Leu 435 440 445 Leu Gly Leu Gly Gly Leu Leu Leu Leu Val Pro Val Ile Tyr Gln Leu 450 455 460 Arg Ser Gln Glu Lys Cys Phe Leu Phe Trp Ser Gly Ser Lys Lys Gly 465 470 475 480 Ser Gln Asp Lys Glu Ala Ile Gln Ala Tyr Ser Glu Ser Leu Met Ser 485 490 495 Pro Ala Ala Lys Gly Thr Val Leu Gln Glu Ala Lys Leu 500 505 2032 base pairs nucleic acid double linear cDNA NO NO misc. feature 40..1926 /Function = “Nucleotides 40 through 1926 encode the amino acid seq 5 GACCGTATCT ATACATTAAG TTCGTAATAT CTCTGCGGAA TGGAATTTTT CTGGACTCTG 60 GCTGTGATTG TGATATATTG TATAGGTCAC ATTCATGGAC GATGTGAAAG ATCTATAGAT 120 TTGGATAATG GAAGTATAAA TTATCGACAG AGAAATATAG TGAGATTCAG ATGCAATCGC 180 GGCTACACTT TGCAGGGAAC AGTAATGCAA ACTTGCGATC GAGATGGTCG CCTTCGAGGC 240 GAAAAACCAT TCTGTGCCAG TAGGGGATGT GCGAGGCCCG AGGATCCGGA GAACGGACAC 300 GTCGAAAATC TTTCCCTAAG GGCGGATGTC GTGTGCCACG ATGGCTATGT CTTGGTCGGT 360 GGTCGCACTG CCTACTGCGA TGGAGAAAGA TGGAGCACCC AGCTGGGATC GTGTCGAAGG 420 AGCAACCACA CAAGAGATCA TTCTTGCGAT TTCGAGAGCG AGGATCAGTG CGGTTGGGAG 480 GCGGAGACAA CCTTCCGACG ACCCTGGAAG CGAGTCAGCA CGGTATCCGA TATTCACTCC 540 CTAAGAACGG GACCCCGCCA CGATCACACG TTTAAAAACG AATCCGGTGG TCATTACATG 600 CGCATGGAAA CCCAAATGGG GGCTTATGGA AGCTACCATC TGCTATCGCC GATCTATCCC 660 AGATCCCTCA CCCTGAAGAC CGCCTGCTGC TTTCGATTCC ACTACTTCAT GTTTGGCGCT 720 GGTGTGGATA ATCTGGTGGT GTCCGTTAAA CCCGTTTCGA TGCCAATGGC AACCATGTGG 780 AATAGGTTCA GAGCCAATTG CAGCAAATTT GAGATATCTG GTCAGCAGGG AACCCAGTGG 840 CTAGAGCACA CGATCAGCAT TGACGAGATG CAAGAGGACT TCCAGGTGAT ATTCACGGCA 900 ACGGATGCAA GATCCCAATT CGGAGATATT GCCATCGATG ATGTAAAGCT AATGACAGGC 960 AGTGAGTGTG GCACAAACGG ATTTAGCACC ACCACAGAAC CAACGGCTCC GACAGGCAGC 1020 AACGAGCAAC CACTGGTCTA CGATATGATA AGTTGTTCAG GTCGATGCGG AACATCAATG 1080 TCGGCCTCCA ATATAACCAA CAATGGTATA GTCATGGGAT GTGGATGTAA TGACGAGTGC 1140 CTTTCGGATG AGACTTGTTG CCTAAACTAT TTGGAGGAGT GCACAAAGGA GCTGCTCACC 1200 ACGACCGAGG ATGATATTAG TTCCCTGCCC CCAACGGTCA CATCAACAAG CACAAGCACT 1260 ACGAGGAAGT CAACAACAAC AACAACCACA AGCACGACTA CTACAAGTAC AACAACAACT 1320 AAAAGGCCAA CCACAACCAC AACAACAACA AAGGCCACAA CTACAAAGCG AACAACAACC 1380 ACTAAAAAAC CGACAACAAC TTCAACAACG CCGAAGCCAA CAACAACGAC TTCAACCACA 1440 CCAAAGTCTA CAACTTCTAC AACGTCTACA ACTTCAACAA CACCAACGAC AACAACTACA 1500 ATAAATGTGT TTACAACAAA GAAAACAACA ATAATGATCC CTACTTCCAG TACCGAAAAG 1560 ACTACAGGCA TCATCACCAC CATGAAGACA CGCAAGCGCA TCACTTGGAA CGTTGATCCT 1620 CAGGACATCG AGGGTCACAT GGACACGAGC GGAAGTACCC CCAATCCAGC TTTAGTAGTA 1680 CTTTACCTGC TACTCGGCAT TGTTCTGGTG GTAGTTCTGG CCAACGTCGT TAATCGCTGG 1740 ATAATACCAA TCACTGGATC AAAGACCAGC AGCGAAAAGG CTGTGAGATT CAAGAAGGCA 1800 TTCGATAGTC TGAAGAAGCA ACGGAAAAGA AACAGCATGG ATGATCAGCC GTTATGCGAC 1860 TCCGATAACG ACGATGTAGA GTATTTCGAA GAAATGGGCG TGGACATACG ACATAGGACC 1920 GATCTATGAG GGTAATCCCC AGTGATACCA AAACAAACGC TTAGGCCTGT GCCTATTGTA 1980 TAGGATGTTT CTAAATGTGT ATGCAAGAAT CGAATAAAAG AAAATATGCA AC 2032 629 amino acids amino acid linear protein NO internal misc. feature 1..629 /Function = ”Amino acid sequence for the Drosophila Melanogaster S Modified-site 30..353 /note= “Positions 30-32, 90-92, 129-131, 180-182, 253-255 and 351-353 represent potential N-glycosylation sites.” Modified-site 1..20 /note= “Amino acids 1-20 represent a putative signal sequence.” Modified-site 21..74 /note= “Amino acids 21-74 represent complement control protein domain number 1.” Modified-site 75..127 /note= “Amino acids 75-127 represent complement control protein domain number 2.” Modified-site 128..312 /note= “Amino acids 128-312 represent an MAM domain.” Disulfide-bond 22..381 /note= “The cysteines at positions 22, 45, 59, 72, 77, 99, 113, 125, 136, 144, 216, 217, 254, 310, 339, 343, 361, 363, 367, 373, 374 and 381 represent potential disulfide linkages.” Modified-site 338..381 /note= “Amino acids 338-381 represent a somatomedin B domain.” Modified-site 387..514 /note= “Amino acids 387-514 represent a mucin-like potential O-linked glycosylation region.” Domain 544..564 /note= “Amino acids 544-565 represent a putative TM domain.” Domain 565..629 /note= “Amino acids 565-629 represent a putative cytoplasmic domain.” Modified-site 576..602 /note= “Amino acids 576-579 and 599-602 represent casein kinase II sites.” Modified-site 578..592 /note= “Amino acids 578-580 and 590-592 represent protein kinase C sites.” Modified-site 596..599 /note= “Amino acids 596-599 represent a cAMP protein kinase site.” 6 Met Glu Phe Phe Trp Thr Leu Ala Val Ile Val Ile Tyr Cys Ile Gly 1 5 10 15 His Ile His Gly Arg Cys Glu Arg Ser Ile Asp Leu Asp Asn Gly Ser 20 25 30 Ile Asn Tyr Arg Gln Arg Asn Ile Val Arg Phe Arg Cys Asn Arg Gly 35 40 45 Tyr Thr Leu Gln Gly Thr Val Met Gln Thr Cys Asp Arg Asp Gly Arg 50 55 60 Leu Arg Gly Glu Lys Pro Phe Cys Ala Ser Arg Gly Cys Ala Arg Pro 65 70 75 80 Glu Asp Pro Glu Asn Gly His Val Glu Asn Leu Ser Leu Arg Ala Asp 85 90 95 Val Val Cys His Asp Gly Tyr Val Leu Val Gly Gly Arg Thr Ala Tyr 100 105 110 Cys Asp Gly Glu Arg Trp Ser Thr Gln Leu Gly Ser Cys Arg Arg Ser 115 120 125 Asn His Thr Arg Asp His Ser Cys Asp Phe Glu Ser Glu Asp Gln Cys 130 135 140 Gly Trp Glu Ala Glu Thr Thr Phe Arg Arg Pro Trp Lys Arg Val Ser 145 150 155 160 Thr Val Ser Asp Ile His Ser Leu Arg Thr Gly Pro Arg His Asp His 165 170 175 Thr Phe Lys Asn Glu Ser Gly Gly His Tyr Met Arg Met Glu Thr Gln 180 185 190 Met Gly Ala Tyr Gly Ser Tyr His Leu Leu Ser Pro Ile Tyr Pro Arg 195 200 205 Ser Leu Thr Leu Lys Thr Ala Cys Cys Phe Arg Phe His Tyr Phe Met 210 215 220 Phe Gly Ala Gly Val Asp Asn Leu Val Val Ser Val Lys Pro Val Ser 225 230 235 240 Met Pro Met Ala Thr Met Trp Asn Arg Phe Arg Ala Asn Cys Ser Lys 245 250 255 Phe Glu Ile Ser Gly Gln Gln Gly Thr Gln Trp Leu Glu His Thr Ile 260 265 270 Ser Ile Asp Glu Met Gln Glu Asp Phe Gln Val Ile Phe Thr Ala Thr 275 280 285 Asp Ala Arg Ser Gln Phe Gly Asp Ile Ala Ile Asp Asp Val Lys Leu 290 295 300 Met Thr Gly Ser Glu Cys Gly Thr Asn Gly Phe Ser Thr Thr Thr Glu 305 310 315 320 Pro Thr Ala Pro Thr Gly Ser Asn Glu Gln Pro Leu Val Tyr Asp Met 325 330 335 Ile Ser Cys Ser Gly Arg Cys Gly Thr Ser Met Ser Ala Ser Asn Ile 340 345 350 Thr Asn Asn Gly Ile Val Met Gly Cys Gly Cys Asn Asp Glu Cys Leu 355 360 365 Ser Asp Glu Thr Cys Cys Leu Asn Tyr Leu Glu Glu Cys Thr Lys Glu 370 375 380 Leu Leu Thr Thr Thr Glu Asp Asp Ile Ser Ser Leu Pro Pro Thr Val 385 390 395 400 Thr Ser Thr Ser Thr Ser Thr Thr Arg Lys Ser Thr Thr Thr Thr Thr 405 410 415 Thr Ser Thr Thr Thr Thr Ser Thr Thr Thr Thr Lys Arg Pro Thr Thr 420 425 430 Thr Thr Thr Thr Thr Lys Ala Thr Thr Thr Lys Arg Thr Thr Thr Thr 435 440 445 Lys Lys Pro Thr Thr Thr Ser Thr Thr Pro Lys Pro Thr Thr Thr Thr 450 455 460 Ser Thr Thr Pro Lys Ser Thr Thr Ser Thr Thr Ser Thr Thr Ser Thr 465 470 475 480 Thr Pro Thr Thr Thr Thr Thr Ile Asn Val Phe Thr Thr Lys Lys Thr 485 490 495 Thr Ile Met Ile Pro Thr Ser Ser Thr Glu Lys Thr Thr Gly Ile Ile 500 505 510 Thr Thr Met Lys Thr Arg Lys Arg Ile Thr Trp Asn Val Asp Pro Gln 515 520 525 Asp Ile Glu Gly His Met Asp Thr Ser Gly Ser Thr Pro Asn Pro Ala 530 535 540 Leu Val Val Leu Tyr Leu Leu Leu Gly Ile Val Leu Val Val Val Leu 545 550 555 560 Ala Asn Val Val Asn Arg Trp Ile Ile Pro Ile Thr Gly Ser Lys Thr 565 570 575 Ser Ser Glu Lys Ala Val Arg Phe Lys Lys Ala Phe Asp Ser Leu Lys 580 585 590 Lys Gln Arg Lys Arg Asn Ser Met Asp Asp Gln Pro Leu Cys Asp Ser 595 600 605 Asp Asn Asp Asp Val Glu Tyr Phe Glu Glu Met Gly Val Asp Ile Arg 610 615 620 His Arg Thr Asp Leu 625 

We claim:
 1. An isolated scavenger receptor protein type BI which selectively binds to low density lipoprotein and to modified lipoprotein having the characteristics of acetylated low density lipoprotein consisting of the sequence shown in Sequence ID No. 4 or the sequence having conservative substitutions, additions and deletions thereof.
 2. The protein of claim 1 having the same amino acid sequence as the protein naturally occurring in cells selected from the group consisting of adipocytes, lung and liver.
 3. The protein of claim 1 encoded by a nucleotide sequence hybridizing under stringent conditions to Sequence ID No.
 3. 4. The protein of claim 3 wherein the sequence is Sequence ID No. 3 or a degenerate variant thereof.
 5. The protein of claim 1 having an amino acid sequence consisting of the sequence shown in Sequence ID No.
 4. 6. The protein of claim 1 immobilized to an inert substrate in a form useful for binding of low density lipoprotein.
 7. The protein of claim 1 of human origin.
 8. The protein of claim 1 expressed on the surface of a cell genetically engineered to express the protein. 